Buprenorphine signalling is compromised at the N40D polymorphism of the human μ opioid receptor in vitro

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1 British Journal of Pharmacology DOI:1.1111/bph RESEARCH PAPER Buprenorphine signalling is compromised at the N4D polymorphism of the human μ opioid receptor in vitro Correspondence Mark Connor, 2 Technology Place, Macquarie University, NSW 219, Australia. mark.connor@mq.edu.au Received 5 March 214 Revised 2 May 214 Accepted 3 May 214 Alisa Knapman, Marina Santiago and Mark Connor Australian School of Advanced Medicine, Macquarie University, Sydney, NSW, Australia BACKGROUND AND PURPOSE There is significant variation in individual response to opioid drugs, which may result in inappropriate opioid therapy. Polymorphisms of the μ opioid receptor (MOP receptor) may contribute to individual variation in opioid response by affecting receptor function, and the effect may be ligand-specific. We sought to determine functional differences in MOP receptor signalling at several signalling pathways using a range of structurally distinct opioid ligands in cells expressing wild-type MOP receptors (MOPr-) and the commonly occurring MOP receptor variant, N4D. EXPERIMENTAL APPROACH MOPr- and MOPr-N4D were stably expressed in CHO cells and in AtT-2 cells. Assays of AC inhibition and ERK1/2 phosphorylation were performed on CHO cells, and assays of K activation were performed on AtT-2 cells. Signalling profiles for each ligand were compared between variants. KEY RESULTS Buprenorphine efficacy was reduced by over 5% at MOPr-N4D for AC inhibition and ERK1/2 phosphorylation. Buprenorphine potency was reduced threefold at MOPr-N4D for K channel activation. Pentazocine efficacy was reduced by 5% for G-protein-gated inwardly rectifying K channel activation at MOPr-N4D. No other differences were observed for any other ligands tested. CONCLUSIONS AND IMPLICATIONS The N4D variant is present in 1 5% of the population. Buprenorphine is a commonly prescribed opioid analgesic, and many individuals do not respond to buprenorphine therapy. This study demonstrates that buprenorphine signalling to several effectors via the N4D variant of MOP receptors is impaired, and this may have important consequences in a clinical setting for individuals carrying the N4D allele. Abbreviations DAMGO, ([D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin); FSK, forskolin; GIRK, G protein-gated inwardly rectifying K channel; MOP receptor, μ-opioid receptor; OPRM1, μ1 opioid receptor gene; PTX, Pertussis toxin 214 The British Pharmacological Society British Journal of Pharmacology (214)

2 A Knapman et al. Table of Links TARGETS μ opioid receptor LIGANDS DAMGO buprenorphine β-endorphin endomorphin-1 endomorphin-2 fentanyl naloxone methadone morphine oxycodone pentazocine This Table lists protein targets and ligands which are hyperlinked to corresponding entries in the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 214) and the Concise Guide to PHARMA- COLOGY 213/14 (Alexander et al., 213). Introduction Opioids are powerful analgesics used for the clinical management of moderate to severe pain. Opioid use is associated with adverse effects such as respiratory depression, nausea, constipation and sedation, and tolerance and dependence can develop with continued opioid use. There is significant variation between individuals in response to opioid drugs, both in the analgesic and adverse effects (Lotsch and Geisslinger, 25). This variation can lead to restricted dosing of opioid analgesics due to the unpredictability of serious adverse events such as respiratory depression, resulting in inadequate pain relief for many individuals (Skorpen et al., 28). The inter-individual variability observed in response to opioids is likely to be caused, at least in part, by genetic differences in proteins responsible for drug absorption, distribution and metabolism, as well as differences in drug/ receptor interactions and receptor signalling (Somogyi et al., 27). A clearer understanding of the genetic factors contributing to individual response to opioids could result in the ability to better predict the outcomes of opioid administration in individuals, leading to more rational choice of drug and dose, thus potentially limiting adverse effects and the development of tolerance and dependence. The μ-opioid receptor (MOP receptor, Cox et al., 214) is a GPCR that is the main site of action for clinically important opioids. The MOP receptor mediates the analgesic and almost all of the adverse effects of these opioid drugs, and it is likely that the genetic variation of OPRM1, the gene coding for MOP receptors, could contribute to individual variation in the response to opioid analgesics. A number of nonsynonymous allelic variants of OPRM1 have been identified within the population, each resulting in an alternative receptor isoform (Lotsch and Geisslinger, 25). The N4D variant is the most common MOP receptor variant, occurring at an allelic frequency of 1 5% in various populations (Mura et al., 213). This variant arises from an A > G substitution at nucleotide 118, resulting in an asparagine to aspartic acid amino acid exchange in the N-terminal domain of MOP receptor, and removing a putative glycosylation site (Singh et al., 1997). Many studies have investigated the association between carriers of MOP receptor-n4d and various clinical outcomes, such as the degree of pain relief from opioid analgesics and the susceptibility to substance abuse. A number of these studies suggest that carriers of the D4 allele require higher doses of post-operative opioid analgesics; however, other studies have shown an increased sensitivity to opioids and a reduced perception of pain (Diatchenko et al., 211; Mura et al., 213). There are also reports of an increased susceptibility to alcohol and heroin abuse in D4 carriers, but an improved response to naltrexone treatment of alcoholism (Mague and Blendy, 21). Despite the volume of research into the effect of the N4D variant on disease outcomes, many of the reports are contradictory and there is no clear consensus as to the effect of the N4D variant on the outcomes of opioid administration or disease susceptibility (Walter and Lotsch, 29). Fewer studies have investigated the consequences of the N4D variant on MOP receptor signalling and function in vitro, and the results of these studies are also conflicting. One study reported a threefold increase in β-endorphin affinity for MOP receptors heterologously expressed in AV-12 cells, and a similar increase in β-endorphin potency for G protein-gated inwardly rectifying K channel (GIRK) activation in Xenopus oocytes (Bond et al., 1998). Subsequent studies have found no differences in β-endorphin potency between N4D- and wildtype ()-MOP receptors (Befort et al., 21; Beyer et al., 24; Kroslak et al., 27). Other studies have reported both increased and decreased DAMGO ([D-Ala2, N-MePhe4, Glyol]-enkephalin) and morphine potency, differences in regulatory processes with chronic opioid treatment, and brain 4274 British Journal of Pharmacology (214)

3 μ opioid receptor N4D SNP signalling BJP region-dependent differences in expression and signalling; however, there is little consistency in these reports (reviewed in Knapman & Connor, 214). As the MOP receptor interacts with many effector and regulatory proteins, changing assay parameters and cellular background may affect MOP receptor signalling in different ways. Furthermore, most studies have used only a single or a small subset of opioid ligands, and may have failed to capture ligand-specific differences in N4D signalling. Like all GPCRs, the MOP receptor has many active conformations, and in the absence of ligand constantly oscillates through a range of possible active and inactive states (Pineyro and Archer-Lahlou, 27; Kenakin and Miller, 21; Manglik et al., 212). Ligand-biased signalling or functional selectivity has been well characterized, where structurally distinct ligands stabilize the receptor in a restricted subset of conformations, preferentially activating certain effectors. The corollary of this phenomenon is that changes in amino acid residues arising from genetic polymorphisms may also affect conformation of the receptor (Abrol et al., 213; Cox, 213). Thus, the N4D variant may affect MOP receptor signalling globally or in a ligand-dependent manner by affecting the ability of a ligand to bind to the receptor, altering the conformation of the ligand-receptor complex and/or affecting the ability of this complex to couple to G-proteins and associated signalling or regulatory pathways. Clinically used opioids are chemically diverse, and are likely to have subtle differences in their interaction with structural features of the MOP receptor, potentially leading to distinct effects of the N4D variant on different drugs. In this study, the ability of human MOPr- and MOPr-N4D to couple to several distinct signalling pathways was investigated in CHO cells and mouse AtT-2 cells using 11 clinically important and/or structurally distinct opioid ligands. We found that the N4D variant had a negative effect on the signalling of the commonly prescribed opioid buprenorphine in all of our assays. In addition, the efficacy of pentazocine was significantly reduced for GIRK activation in AtT-2 cells expressing N4D. No differences in signalling via other opioids, including β-endorphin, were observed. Methods MOP receptor transfection and cell culture CHO-FRT-TRex cells were stably transfected with a pcdna5 construct encoding the haemagglutinin (HA)-tagged human μ-opioid receptor cdna together with the pog44 (Flp recombinase plasmid) using the transfectant Fugene (Promega), as described in previously (Knapman et al., 214). The HA-tagged human wild-type μ-opioid receptor (MOPr-) and the μ-opioid receptor containing the D4 variant (MOPr- N4D) were synthesized by Genscript (Piscataway, NJ, USA). Cells expressing MOPr- or MOPr-N4D were selected using hygromycin B (5 μg ml 1 ). Cells were cultured in DMEM containing 1% FBS, 1 U penicillin/streptomycin and 5 μg ml 1 hygromycin B up to passage 5. Hygromycin concentration was reduced to 2 μg ml 1 beyond passage 5. AtT-2 FlpIn cells were constructed by transfecting Flprecombinase target site (FRT)/LacZeo2 (Life Technologies, Mulgrave, VIC, Australia) using Fugene. Successfully transfected cells were selected with 1 μg ml 1 zeocin, and stably transfected with MOPr- or MOPr-N4D as described for CHO cells. Cells expressing MOP receptors were selected using 1 μg ml 1 hygromycin B. Cells were cultured in DMEM containing 1% FBS, 1 U penicillin/streptomycin and 1 μg ml 1 hygromycin B. CHO-MOP receptor cells and AtT-2-MOP receptor cells were passaged at 8% confluency as required. Assays were carried out on cells up to 3 passages. Cells for assays were grown in 75 cm 2 flasks and used at greater than 9% confluence. MOP receptor expression Surface expression of MOP receptors was determined on intact CHO-MOP receptor cells by incubation with nm [ 3 H]-DAMGO (D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin; PerkinElmer, Waltham, MA, USA) at 4 C in 5 mm Tris-Cl (ph 7.4) for 2 h. Briefly, 24 h before the assay, cells were detached from flasks with trypsin/edta (Sigma, Castle Hill, NSW, Australia) and resuspended in DMEM containing 1% FBS, 1 U penicillin/streptomycin, plus 2 μg ml 1 tetracycline to induce MOP receptor expression. Cells were seeded at a density of cells per well in 24-well plates pre-coated with polylysine and grown overnight at 37 C. On the day of the assay, cells were washed twice gently with 5 mm Tris-Cl (ph 7.4) and incubated for 2 h on ice with nm [ 3 H]-DAMGO. Non-specific binding was determined in the presence of unlabelled DAMGO (1 μm). Non-specific binding was 15 ± 1% of total binding for CHO-MOPr-, and 11 ± 1% in CHO-MOPr- N4D. At the end of the incubation, plated cells were washed three times with 5 mm Tris-Cl (ph 7.4) at 4 C. Cells in each well were then digested for 1 h at RT with 1 μl of 1N NaOH. Then, 1 μl 1N HCl was added to each well and collected into scintillation vials and bound ligand determined using a liquid scintillation counter (Packard Tricarb, Perkin Elmer). Receptor density (B max) and affinity (K D) were calculated using a one-site binding curve fitted using GraphPad Prism (Graph- Pad Software, La Jolla, CA, USA). Protein concentration was determined with a BCA Protein Assay Kit (Pierce) according to the manufacturer s instructions. All experiments were performed three times in triplicate. Membrane potential assay of AC inhibition Opioid inhibition of AC was measured using an assay of membrane potential (Knapman et al., 214). The day before the assay, CHO-MOP receptor cells were detached from the flask with trypsin/edta (Sigma) and resuspended in 1 ml of Leibovitz s L-15 media supplemented with 1% FBS, 1 U penicillin/streptomycin and 15 mm glucose. MOP receptor expression was induced with 2 μg ml 1 tetracycline 24 h before the assay. The cells were plated in a volume of 9 μl in black-walled, clear-bottomed 96-well microplates (Corning, Tewksbury, MA, USA), and incubated overnight at 37 C in ambient CO 2. Membrane potential was measured using a FLIPR Membrane Potential Assay kit (blue) from Molecular Devices (Sunnyvale, CA, USA). The dye was reconstituted with assay buffer (HBSS) containing (in mm) NaCl 145, HEPES 22, Na 2HPO 4.338, NaHCO , KH 2PO 4.441, MgSO 4.47, MgCl 2.493, CaCl , glucose 5.56 (ph 7.4, British Journal of Pharmacology (214)

4 A Knapman et al. osmolarity 315 ± 5). Prior to the assay, cells were loaded with 9 μl per well of the dye solution without removal of the L-15, giving an initial assay volume of 18 μl per well. Plates were then incubated at 37 C in ambient CO 2 for 6 min. Fluorescence was measured using a FlexStation 3 (Molecular Devices) microplate reader with cells excited at a wavelength of 53 nm and emission measured at 565 nm. Baseline readings were taken every 2 s for at least 2 min, at which time forskolin (FSK, an activator of AC) and either opioid or vehicle was added in a volume of 2 μl. The background fluorescence of cells without dye or dye without cells was negligible. Changes in fluorescence were expressed as a percentage of baseline fluorescence after subtraction of the changes produced by vehicle addition. The final concentration of DMSO was not more than.1%, and this concentration did not produce a signal in the assay. Membrane potential assay of GIRK channel activation Opioid activation of endogenous GIRK channels in AtT-2 cells was measured using a membrane potential sensitive dye, as previously described (Knapman et al., 213). AtT-2-MOP receptor cells were detached from flasks and plated using the same procedure as for the CHO-MOP receptor cells in the AC inhibition assay, with no addition of tetracycline. Blue membrane potential dye was reconstituted and loaded onto cells, and the assay was performed in the same way as the AC inhibition assay. Baseline readings were taken every 2 s for at least 2 min, at which time opioid or vehicle was added in a volume of 2 μl. Measurements were taken at the peak decrease in signal from baseline, approximately 1 2 s after drug addition. The background fluorescence of cells without dye or dye without cells was negligible. The final concentration of DMSO was not more than.1%, and this concentration did not produce a signal in the assay. ELISA of ERK1/2 phosphorylation Opioid-induced phosphorylation of ERK1/2 was measured by ELISA. The day before the assay, CHO-MOP receptor cells were plated and receptor expression was induced as for the membrane potential assay. Cells were plated in 96-well clear microplates (Falcon, Tewksbury, MA, USA). On the day of the assay, cells were deprived of serum for 1 h in 4 μl serum-free L-15 supplemented with 5% BSA. All cell treatments were in a volume of 4 μl unless stated otherwise. Serum-deprived cells were treated with drug or vehicle diluted in serum-free L-15. Preliminary experiments indicated that drug treatment for 5 min was optimal to produce robust ERK1/2 phosphorylation without inducing desensitization, thus all drug treatments were for 5 min unless otherwise indicated. After drug application, the reaction was stopped by inverting the plates to remove the drug solution, placing the plates on ice and immediately fixing the cells with 4% paraformaldehyde for 15 min at room temperature (RT). Cells were washed three times with 3 μl PBS, then permeabilized with.1% Triton-X in PBS for 3 min at RT. Triton-X was removed, and cells were incubated for 2 h at RT with blocking solution consisting of 5% BSA in PBS with.1% Tween-2 (PBS-T). Blocking solution was removed, then cells were incubated overnight at 4 C with a 1:5 dilution of rabbit antiphospho-p44/42 MAPK (Thr 22 /Tyr 24 ) antibody in PBS-T with 1% BSA. Cells were washed three times with 3 μl PBS-T, and incubated with 1:5 anti-rabbit IgG HRP-linked antibody in PBS-T with 1% BSA for 2 h. Cells were washed four times with 3 μl PBS-T, and incubated with 3,3,5,5 - tetramethylbenzidine (Sigma) at RT in the dark for 45 min. The reaction was stopped with 1M HCl. Absorbance was read at 45 nm using a BMG Pherastar FS microplate reader (BMG Labtech, Mornington, VIC, Australia). Cells were then stained with.5 μg ml 1 DAPI for 1 min at RT, and washed three times with 3 μl PBS-T. Fluorescence was read in the Pherastar microplate reader with cells excited at a wavelength of 358 nm and emission measured at 461 nm. Absorbance readings were normalized to DAPI staining to account for differences in cell density between wells. Readings were then normalized to the response of cells treated with 1 nm phorbol 12-myristate 13-acetate (PMA) for 1 min. Drugs and chemicals Unless otherwise stated, tissue culture reagents and buffer salts were from Life Technologies or Sigma. DAMGO, endomorphin-1, endomorphin-2 and met-enkephalin were purchased from Auspep (Tullamarine, Australia). Morphine and pentazocine were a kind gift from the Department of Pharmacology, University of Sydney. Buprenorphine and oxycodone were from the National Measurement Institute (Lindfield, Australia). β-endorphin was from Genscript. Fentanyl (Andrews Laboratories) was a gift from the Department of Pharmacology, Sydney University. FSK and naloxone were from Ascent Pharmaceuticals (Bristol, UK). 1,9- Dideoxyforskolin and tetraethylammonium (TEA) were from Sigma Aldrich (Castle Hill, Australia). Pertussis toxin (PTX) was from Tocris Bioscience (Bristol, UK). Phospho-ERK1/2 antibody (Catalogue #911) and anti-rabbit IgG HRP-lined antibody (Catalogue #774) were from Cell Signaling Technologies, Danvers, MA, USA. Data Unless otherwise noted, data are expressed as mean ± SEM of at least five determinations made in duplicate or triplicate. Concentration-response curves were fit with a four parameter logistic equation using Graphpad Prism (Graphpad). E max and pec 5 values were derived from individual experiments and compared using Student s unpaired t-test. P <.5 was considered significant. Full agonist activity was determined by comparing E max values derived from individual experiments using one-way ANOVA, corrected for multiple comparisons. Pooled data from replicate experiments are shown in the Figures for ease of reference. All channel and receptor nomenclature is consistent with the British Journal of Pharmacology/IUPHAR Concise Guide to Pharmacology (Alexander et al., 213). Results MOP receptor expression in CHO-K1 cells CHO-K1 cells were stably transfected with either MOPr- or the MOPr-N4D variant, with receptor expression controlled by a tetracycline-sensitive repressor. After induction of MOP receptor expression with tetracycline, specific binding of [ 3 H]- DAMGO was similar for both variants, indicating similar 4276 British Journal of Pharmacology (214)

5 μ opioid receptor N4D SNP signalling BJP Specific Binding (DPM) [( 3 H)DAMGO] nm N4D Figure 1 Saturation binding curve of [ 3 H]-DAMGO in intact CHO-MOPr- and CHO-MOPr-N4D cells, 24 h after induction of receptor expression with tetracycline. Radioligand binding was carried out as described in the Methods. No significant difference in B max or K D was observed between cells expressing MOPr- or MOPr-N4D (P >.5). Each point represents the mean ± SEM of triplicate determinations from a single experiment. The assay was repeated three times. Figure 2 DAMGO inhibits AC and activates ERK1/2 in CHO cells expressing MOPr- or MOPr-N4D. AC inhibition and levels of ERK1/2 phosphorylation were determined as described in the Methods. (A) Traces showing changes in fluorescent signal following application of 3 nm FSK + vehicle (HBSS) to MOPr-, and 3 nm FSK + 1 μm DAMGO to MOPr- and MOPr-N4D cells. Drugs were added at 12 s. Changes in raw fluorescent units (RFU) are normalized to predrug values. (B) DAMGO inhibited FSK-stimulated AC hyperpolarization of MOPr- or MOPr-N4D to a similar degree and with a similar potency (P >.5). (C) DAMGO stimulated ERK1/2 phosphorylation in cells expressing MOPr- or MOPr-N4D to a similar degree and with a similar potency (P >.5). Maximum ERK1/2 phosphorylation via 1 nm PMA was used as a control for perk1/2 experiments. Data represent the mean ± SEM of pooled data from five to six independent determinations performed in duplicate. A RFU (% baseline) B C nm FSK + HBSS 3 nm FSK + 1 µm DAMGO () 3 nm FSK + 1 µm DAMGO (N4D) (%) N4D Time (s) [DAMGO] log M levels of surface receptor expression (see Figure 1). The B max for CHO-MOPr- cells was 28 ± 2 fmol mg 1 total protein and 356 ± 18 fmol mg 1 for CHO-MOPr-N4D (P >.5). The affinity for [ 3 H]-DAMGO was also similar between for -MOPr and N4D-MOPr expressing cells, with K D of.75 ±.1 and.65 ±.2 respectively (P >.5). AC inhibition In CHO-MOP receptor cells loaded with membrane potential dye, addition of the AC activator FSK (3 nm) produced a rapid decrease in fluorescence, consistent with hyperpolarization of the cells (Figure 2A; Knapman et al., 214). The hyperpolarization stabilized 5 min after the addition of FSK, at which point measurements were taken. FSK (3 nm) produced a similar hyperpolarization in CHO-MOPr- (42 ± 1% decrease in fluorescence) and CHO-MOPr-N4D (44 ± 2% N4D [DAMGO] log M British Journal of Pharmacology (214)

6 A Knapman et al. Table 1 Summary of opioid efficacy and potency in assays of AC inhibition in CHO cells expressing MOPr- and MOPr-N4D AC inhibition E max (%) pec 5 Opioid N4D N4D β-endorphin 7 ± 4 69 ± ± ±.1 Methadone 66 ± 2 73 ± 5 7. ± ±.6 Met-enkephalin 66 ± 4 73 ± ± ±.3 Morphine 66 ± 3 7 ± 4 7. ±.1 7. ±.1 Fentanyl 65 ± 2 74 ± ± ±.1 Oxycodone 65 ± 5 77 ± ±.3 6. ±.1 Endomorphin-1 62 ± 3 61 ± ± ±.1 Endomorphin-2 6 ± 7 64 ± ± ±.1 DAMGO 6 ± 2 67 ± ± ±.2 Pentazocine 5 ± 6 47 ± ± ±.6 Buprenorphine 35 ± 6 16 ± 4* 8.5 ±.2 8. ±.2 Assays were performed as described in the Methods. Opioids are listed in rank order of maximal effect at MOPr-. Opioids with E max significantly lower than methadone are set in bold (one-way ANOVA, followed by Student s t-test, corrected for multiple comparisons, P <.5). *E max or pec 5 significantly different between MOPr- and MOPr-N4D (P <.5). decrease) cells. (P >.5). Simultaneous addition of the prototypical MOP receptor selective peptide agonist DAMGO with FSK resulted in a concentration-dependent inhibition of the FSK-stimulated hyperpolarization in both cell lines (Figure 2). DAMGO maximally inhibited the FSK response by 6 ± 2%, with pec 5 of 7.7 ±.1 in CHO-MOPr- cells (Table 1). DAMGO inhibited FSK similarly in cells expressing N4D, E max was 67 ± 6% and pec ±.1 (P >.5). We have reported previously that the effects of DAMGO in this assay were sensitive to naloxone and strongly inhibited by overnight pretreatment with PTX (Knapman et al., 214). Application of opioids alone did not affect the membrane potential of CHO-MOP receptor cells with the exception of 1 μm methadone and 3 μm pentazocine, which both caused a small, transient increases in fluorescence (<1%). These effects were not naloxone-sensitive and were not observed at lower concentrations of drug. They are consistent with previously reported inhibition of K channels by these drugs (Nguyen et al., 1998; Matsui and Williams, 21). Because of the uncertainty surrounding the effects of N4D polymorphism on opioid actions, we measured the potency and efficacy of a range of clinically important and/or structurally distinct opioid ligands in CHO cells expressing -MOP receptor or the N4D variant to determine whether the N4D amino acid substitution could affect MOP receptor signalling in a ligand-selective manner. The endogenous opioid β-endorphin has previously been shown to have a threefold increase in potency at GIRK channel activation in Xenopus oocytes expressing N4D-MOPr (Bond et al., 1998). In the present assay, there was no difference in β-endorphin potency or efficacy between CHO-MOPr- cells and CHO- MOPr-N4D cells (Figure 3, Table 1). AC inhibition by the putative endogenous opioid ligands endomorphin-1 and endomorphin-2, as well as met-enkephalin was also similar in cells expressing MOPr- or MOPr-N4D receptor (Figure 3, Table 1). The efficacy and potency of the prototypical opioid alkaloid morphine was not different in cells expressing the N4D variant (see Figure 4, Table 1). Interestingly, the semisynthetic morphine derivative buprenorphine had a significantly lower efficacy for AC inhibition in CHO cells expressing MOPr-N4D when compared with MOPr-. The buprenorphine E max was 35 ± 6% in CHO-MOPr- cells and 16 ± 4% in CHO-MOPr-N4D cells, a reduction of over 5% (P <.5). Buprenorphine potency was similar at the N4D variant (see Figure 4, Table 1). The low efficacy of buprenorphine at MOP receptors could have accentuated a general difference in transduction efficiency between MOPr- and the MOPr- N4D. However, the low efficacy MOP receptor agonist pentazocine inhibited AC to a similar degree in cells expressing MOPr- or MOPr-N4D (Figure 4, Table 1), indicating that the low efficacy of buprenorphine per se was not responsible for reduced signalling at the N4D variant. No difference in AC signalling was observed between cells expressing MOPr- or the MOPr-N4D variant when challenged with fentanyl, methadone or oxycodone (Figure 5, Table 1). Opioid-mediated phosphorylation of ERK1/2 We next examined whether the N4D amino acid substitution leads to alterations in signalling through another important pathway activated by MOP receptors, stimulation of ERK1/2 phosphorylation by MOP receptors. We analysed the ability of opioids to stimulate ERK1/2 phosphorylation via MOPr- and MOPr-N4D in CHO cells, using a whole-cell ELISA assay. Opioid responses were normalized against 1 nm PMA applied for 1 min. The average PMA response was similar in cells expressing MOPr- or MOPr-N4D, with corrected absorbance readings of.56 ±.7 and.6 ±.7 respectively (P >.5). DAMGO-stimulated ERK1/2 phosphorylation was similar between - and N4D-expressing cells, with a 4278 British Journal of Pharmacology (214)

7 μ opioid receptor N4D SNP signalling BJP A (%) 1 B N4D 8 6 N4D [β-endorphin] log M 1 (%) N4D N4D [β-endorphin] log M [Endomorphin-1] log M 1 (%) 8 N4D [Endomorphin-1] log M N4D [Endomorphin-2] log M [Endomorphin-2] log M 1 (%) N4D N4D [Met-Enkephalin] log M [Met-Enkephalin] log M Figure 3 Endogenous opioids inhibit AC and activate ERK1/2 in CHO cells expressing MOPr- or MOPr-N4D. AC inhibition and levels of ERK phosphorylation were determined as described in the Methods. (A) β-endorphin, endomorphins-1 and -2, and met-enkephalin inhibited FSK-stimulated AC hyperpolarization of MOPr- or MOPr-N4D to a similar degree and with a similar potency (P >.5). (B) β-endorphin, endomorphi-1 and -2, and met-enkephalin stimulated ERK1/2 phosphorylation in cells expressing MOPr- or MOPr-N4D to a similar degree and with a similar potency (P >.5). Maximum ERK1/2 phosphorylation via 1 nm PMA was used as a control for perk1/2 experiments. Data represent the mean ± SEM of pooled data from five to six independent determinations performed in duplicate. British Journal of Pharmacology (214)

8 A Knapman et al. A 1 (%) B N4D 8 6 N4D [Morphine] log M [Morphine] log M 1 (%) N4D 8 6 N4D [Buprenorphine] log M [Buprenorphine] log M 1 (%) N4D 8 6 N4D [Pentazocine] Log M [Pentazocine] Log M 4 Figure 4 Buprenorphine inhibits AC and activates ERK1/2 less effectively in CHO cells expressing MOPr-N4D. AC inhibition and levels of ERK phosphorylation were determined as described in the Methods. (A) Buprenorphine E max for inhibition of FSK-stimulated AC hyperpolarization was decreased from 35 ± 6 % in CHO-MOPr- to 16 ± 4% in CHO-MOPr-N4D (P <.5). Morphine and pentazocine inhibited AC hyperpolarization to a similar degree and with similar potency in CHO cells expressing MOPr- and MOPr-N4D (P >.5). (B) Buprenorphine E max for stimulation of ERK1/2 phosphorylation was decreased from 35 ± 7 % in CHO-MOPr- to 14 ± 6% in CHO-MOPr-N4D (P <.5). Morphine and pentazocine stimulated ERK1/2 phosphorylation to a similar degree and with similar potency in CHO cells expressing MOPr- and MOPr-N4D (P >.5). Maximum ERK1/2 phosphorylation via 1 nm PMA was used as a control for perk1/2 experiments. Data represent the mean ± SEM of pooled data from five to six independent determinations performed in duplicate. pec 5 of 8.5 ±.1 and E max of 67 ± 6% of the PMA response in CHO-MOPr- cells and a pec 5 of 8.5 ±.7 and E max of 72 ± 5% in CHO-MOPr-N4D (Figure 2, Table 2). Pre-incubation of cells with 1 μm naloxone for 5 min blocked the response for all opioids tested. Application of DAMGO to cells treated overnight with 2 ng ml 1 PTX or in cells where receptor expression had not been induced did not elicit a response. The ERK1/2 phosphorylation elicited by β-endorphin stimulation of MOPr-N4D was similar to that of MOPr-. β-endorphin E max and pec 5 were 72 ± 2% and 7.5 ±.1, respectively, in CHO-MOPr- cells, and 79 ± 9% and 7.6 ±.1, respectively, in CHO-MOPr-N4D cells (P >.5). Similarly, no differences in potency or efficacy were observed between MOP receptor variants for endomorphin-1, endomorphin-2 and met-enkephalin (Figure 3, Table 2). Buprenorphine efficacy was significantly decreased at the N4D variant. Maximum buprenorphine stimulated ERK1/2 phosphorylation in CHO-MOPr- was 35 ± 7%, while in 428 British Journal of Pharmacology (214)

9 μ opioid receptor N4D SNP signalling BJP A 12 (%) B N4D 1 8 N4D [Fentanyl] log M [Fentanyl] log M 1 (%) N4D 8 6 N4D [Oxycodone] log M [Oxycodone] log M 1 (%) N4D N4D [Methadone] log M [Methadone] log M Figure 5 Fentanyl, oxycodone and methadone inhibit AC and activate ERK1/2 in CHO cells expressing MOPr- or MOPr-N4D. AC inhibition and levels of ERK phosphorylation were determined as described in the Methods. (A) Fentanyl, oxycodone and methadone inhibited FSK-stimulated AC hyperpolarization of MOPr- or MOPr-N4D to a similar degree and with a similar potency (P >.5). (B) Fentanyl, oxycodone and methadone stimulated ERK1/2 phosphorylation in cells expressing MOPr- or MOPr-N4D to a similar degree and with a similar potency (P >.5). Maximum ERK1/2 phosphorylation via 1 nm PMA was used as a control for perk1/2 experiments. Data represent the mean ± SEM of pooled data from five to six independent determinations performed in duplicate. CHO-MOPr-N4D efficacy was 14 ± 6%, a reduction of approximately 6% (P <.5). Buprenorphine potency did not differ between cells expressing MOPr- and MOPr- N4D, with pec 5s of8.7±.4 and 9.3 ±.1 respectively (Figure 4, Table 2). The partial agonists morphine and pentazocine stimulated ERK1/2 phosphorylation similarly at MOPr- and MOPr-N4D (Figure 4, Table 2). Fentanyl-, methadone- and oxycodone-stimulated ERK1/2 phosphorylation was also similar in efficacy and potency between CHO- MOPr- and CHO-MOPr-N4D cells (Figure 5, Table 2). There were minor differences in relative agonist efficacy between MOPr- and MOPr-N4D. In assays of AC inhibition, endomorphin-1, endormorphin-2, pentazocine and buprenorphine had a significantly lower E max than β- endorphin for both MOPr- and MOPr-N4D. DAMGO had a significantly lower E max than β-endorphin to inhibit AC in MOPr- but not in MOPr-N4D. In assays of ERK1/2 phosphorylation in both MOPr- and MOPr-N4D, DAMGO, β-endorphin, pentazocine and buprenorphine had significantly lower E max than the most efficacious agonist in British Journal of Pharmacology (214)

10 A Knapman et al. Table 2 Summary of opioid efficacy and potency in assays of ERK1/2 phosphorylation in CHO cells expressing MOPr- and MOPr-N4D perk1/2 E max (%) pec 5 Opioid N4D N4D Endomorphin-2 19 ± ± ± ±.1 Met-enkephalin 13 ± 9 87 ± ± ±.3 Oxycodone 11 ± 8 19 ± ± ±.1 Fentanyl 99 ± ± ± ±.2 Endomorphin-1 93 ± 6 92 ± ± ±.1 Methadone 88 ± 9 72 ± ± ±.1 β-endorphin 72 ± 9 79 ± ± ±.1 DAMGO 67 ± 6 72 ± ± ±.7 Morphine 65 ± 7 76 ± 7 7. ± ±.1 Pentazocine 39 ± ± ±.2 6. ±.6 Buprenorphine 35 ± 7 14 ± 6* 8.7 ± ±.1 Assays were performed as described in the Methods. Opioids are listed in rank order of maximal effect at MOPr-. Opioids with E max significantly lower than endomorphin-2 are set in bold (one-way ANOVA, followed by Student s t-test, corrected for multiple comparisons, P <.5). *E max or pec 5 significantly different between MOPr- and MOPr-N4D (P <.5). this assay, endomorphin-2. Morphine had a significantly lower E max than endomorphin 2 to stimulate ERK1/2 phosphorylation in MOPr- but not in MOPr-N4D. As there was no statistically significant difference between variants for E max of methadone, endomorphin-2, DAMGO or morphine in any of our assays, presumably the variance inherent in the AC inhibition and ERK1/2 phosphorylation assays precluded the statistical differentiation of small differences in efficacy between ligands. GIRK channel activation in AtT-2 cells Activation of MOP receptors results in activation of GIRK channels via Gβγ subunits. As CHO-K1 cells do not express endogenous GIRK channels, we assessed opioid-mediated GIRK activation in AtT-2 cells stably transfected with hmopr- and hmopr-n4d as previously described (Knapman et al., 213). In AtT-2 cells loaded with membrane potential-sensitive dye, application of opioids resulted in a concentration-dependent decrease in fluorescence from baseline, corresponding to membrane hyperpolarization from GIRK activation (Figure 6). Sub-maximal membrane hyperpolarization produced by low-efficacy agonists such as buprenorphine or low concentrations of high-efficacy agonists such as DAMGO rapidly returned towards baseline within 2 min after addition; however, maximum membrane hyperpolarization produced by high concentrations of highefficacy agonists remained steady over 5 min (Figure 6). There was no difference in maximum DAMGO-stimulated GIRK activation between AtT2-MOPr- cells and AtT2-MOPr- N4D cells, with E max of 34 ±.1% and 32 ±.1% decrease from baseline, respectively (P >.5), and pec 5 for both variants was 8.4 ±.1 (Figure 7, Table 3). Buprenorphine was less potent for GIRK activation in AtT2-MOPr-N4D cells, with pec 5 of 6.7 ±.8 compared with pec 5 of 7. ±.7 in AtT2-MOPr- cells (P <.5). RFU (% baseline) HBSS 3nMDAMGO 3 µm Buprenorphine 3 nm DAMGO Time (s) Figure 6 DAMGO causes membrane hyperpolarization in AtT-2 cells expressing MOPr-. Raw trace showing decrease in fluorescent signal following application of vehicle (HBSS), 3 nm DAMGO, 3 nm DAMGO or 3 μm buprenorphine to AtT2 cells expressing MOPr-, corresponding to membrane hyperpolarization from GIRK activation. The Y-axis is raw fluorescent units (RFU). Drugs were added for the duration of the bar. The traces are representative of at least six individual experiments, each performed in duplicate. Buprenorphine efficacy was unaffected by the N4D variant, MOPr- E max was 22 ± 2%, and MOPr-N4D E max was 2 ± 2% (P >.5). We also observed a significant decrease in the efficacy of the partial agonist pentazocine at MOPr-N4D, the pentazocine E max was 8 ± 1% at MOPr-, and 4 ± 1% at N4D although the pec 5 was similar between variants (Figure 7, Table 3). Morphine, methadone and β-endorphin signalling was unaffected at the N4D variant (Figure 7, 4282 British Journal of Pharmacology (214)

11 μ opioid receptor N4D SNP signalling BJP A Fluorescence (%) N4D B Fluorescence (%) N4D [Buprenorphine] log M [Pentazocine] Log M C Fluorescence (%) N4D Fluorescence (%) 5 4 N4D [DAMGO] log M [Morphine] log M Fluorescence (%) 5 4 N4D Fluorescence (%) 5 4 N4D [ -Endorphin] log M [Methadone] log M Figure 7 Buprenorphine is less potent at activating GIRK in AtT2 cells expressing MOPr-N4D than in those expressing MOPr-. GIRK activation was determined as described in the Methods. (A) Buprenorphine activated GIRK channels to a similar degree in AtT2 cells expressing MOPr- and MOPr-N4D, but with a threefold lower pec 5, from 7. ±.1 in AtT2-MOPr- to 6.7 ±.1 in AtT2-MOPr-N4D (P <.5). (B) Pentazocine activated GIRK channels with 5% lower efficacy at MOPr-N4D, E max was decreased from 8 ± 1% in AtT2-MOPr- to 4 ± 1% in AtT2-MOPr-N4D (P <.5). (C) DAMGO, morphine, β-endorphin and methadone activated GIRK channels to a similar degree and with similar potency in AtT-2 cells expressing MOPr- and MOPr-N4D (P >.5). Data represent the mean ± SEM of pooled data from five to six independent determinations performed in duplicate. Table 3). As buprenorphine efficacy was similar between variants, and significantly less than higher efficacy agonists, it is unlikely that the difference in pentazocine efficacy is due to lower receptor expression in AtT2-MOPr-N4D cells. Similarly, the partial agonist activity of buprenorphine precludes the possibility of a change in receptor reserve contributing to decreased potency for GIRK activation. Discussion and conclusions We have shown that the commonly occurring MOP receptor variant N4D is less effectively activated by buprenorphine in assays of AC inhibition, ERK phosphorylation and GIRK activation. Buprenorphine efficacy was reduced by over 5% for AC inhibition and ERK1/2 phosphorylation in CHO-MOPr British Journal of Pharmacology (214)

12 A Knapman et al. Table 3 Summary of opioid efficacy and potency in assays of GIRK activation in AtT-2 cells expressing MOPr- and MOPr-N4D GIRK activation E max (%) pec 5 Opioid N4D N4D DAMGO 34 ± 1 33 ± ± ±.1 β-endorphin 33 ± 2 34 ± ± ±.1 Methadone 33 ± 2 33 ± ± ±.1 Morphine 31 ± 1 3 ± ± ±.1 Buprenorphine 22 ± 1 2 ± 2 7. ± ±.1* Pentazocine 8 ± 1 4 ± 1* 6.3 ±.6 7. ±.2 Assays were performed as described in the Methods. Opioids are listed in rank order of maximal effect at MOPr-. Opioids with E max significantly lower than DAMGO are set in bold (one-way ANOVA, followed by Student s t-test, corrected for multiple comparisons, P <.5). *E max or pec 5 significantly different between MOPr- and MOPr-N4D are marked with (P <.5, uncorrected for multiple comparisons). cells, with no effect on potency. Buprenorphine efficacy for GIRK activation was not affected by the N4D variant when expressed in AtT-2 cells, but potency was decreased threefold. In assays of GIRK activation, pentazocine efficacy was also significantly decreased, with no change in potency. No other opioids were affected by the N4D variant in any of our assays. Buprenorphine is a partial agonist, and as such, even modest differences in levels of receptor expression between variants and/or the presence of spare receptors could have a significant impact on its signalling profile. Studies in animal models, post-mortem human brain and heterologous expression systems have suggested the N4D variant causes decreased receptor expression, although reports are inconsistent (Zhang et al., 25; Kroslak et al., 27; Mague et al., 29; Oertel et al., 212). MOP receptor expression was similar for both variants in the cell lines used in our assays. The N4D variant causes the removal of a putative N-linked glycosylation site, which has been shown to result in decreased MOP receptor glycosylation and stability in CHO cells (Huang et al., 212). In our study, MOP receptor expression in CHO cells was acutely induced prior to experiments, minimizing possible effects on receptor turnover. For AtT-2 cells, receptor expression was not directly measured; however, the use of the FlpIn system for receptor transfection ensures that the receptor construct is inserted only once into the same location in the genome, thus all cells are subjected to a similar transcriptional environment (Sauer, 1994). Additionally, buprenorphine efficacy was similar in cells expressing MOPr- and MOPr-N4D, suggesting similar expression levels. Our findings suggest that the amino acid change on the N-terminus of the MOP receptor may affect the ability of some opioid ligands to effectively transduce signals to the intracellular effectors of MOP receptor. GPCRs constantly oscillate through a range of possible conformations, and different ligands can more effectively stabilize a subset of receptor conformations (Kenakin and Miller, 21). GPCRs undergo further conformational changes upon ligand binding, and the resulting conformation of the ligand receptor complex may couple differentially to associated G-proteins (Pineyro and Archer-Lahlou, 27). The involvement of the N-terminal domain in MOP receptor conformational changes is not yet well understood, and this region of the receptor is not included in published crystal structures (Manglik et al., 212). However, activation-dependent changes in this region have been observed in MOP receptor and other GPCRs. Antibodies generated against the region proximal to N4D on the N-terminal domain of MOP receptor show differential recognition of activated receptors, suggesting this region undergoes conformational changes upon ligand-binding (Gupta et al., 27; 28). This effect was not seen in cells expressing MOPr-N4D or in cells treated with deglycosylating agents, indicating that agonist binding induces movement of N-glycan chains (Gupta et al., 28). Furthermore, N-terminal antibody binding was affected by changes in the C-terminal region of the MOP receptor, implying that conformational changes associated with receptor coupling to associated G-proteins may influence all domains of the MOP receptor. A similar effect was seen in other GPCRs examined in this study, including the δ-opioid receptor and the cannabinoid CB 1 receptor (Gupta et al., 27). N-terminal polymorphisms affect signalling in other related GPCRs. N-terminal single nucleotide polymorphisms (SNPs) in the 5-HT 2B receptor increased constitutive and agonist-stimulated activity in COS-7 cells (Belmer et al., 214). Likewise, polymorphisms of the N-terminal region in the melanocortin MC 4 receptor increased constitutive activity (Srinivasan et al., 24). Taken together, these results indicate the N-terminal region can undergo substantial structural changes upon receptor activation, and that structural changes arising from genetic variation have the ability to significantly affect receptor function. Thus, the N4D substitution may affect the ability of a ligand to bind to MOP receptors, or affect the efficacy of ligand-directed MOP receptor coupling to effector pathways due to altered receptor conformation. It is not immediately apparent why buprenorphine efficacy is affected in CHO cells while buprenorphine potency changes in AtT-2 cells, but the differences may be due to different effectors. MOP receptor inhibition of AC is mediated by Gα i/o subunits of the G-protein heterotrimer, whereas GIRK activation occurs via Gβγ subunits (Law et al., 2) British Journal of Pharmacology (214)

13 μ opioid receptor N4D SNP signalling BJP Phosphorylation of ERK1/2 can occur via multiple pathways, and may involve Gα and Gβγ-coupled processes as well as G-protein independent pathways such as β-arrestin (Luttrell, 25). Whereas AC inhibition is mediated by a single Gα subunit, GIRK channels require the binding of four Gβγ subunits for activation (Corey and Clapham, 21). Differences in the ability of the N4D variant to couple to various G-proteins may differentially affect opioid ligand signalling at Gα and Gβγ-mediated pathways (Allouche et al., 1999; Galandrin et al., 28). Furthermore, cell lines vary in the available pool of G-proteins, effector molecules and regulatory proteins, and opioids may activate a different suite of G-proteins in CHO cells and AtT-2 cells (Atwood et al., 211). Alternatively, buprenorphine may show functional selectivity towards GIRK activation over AC inhibition or ERK1/2 phosphorylation, although studies of AC inhibition and ERK1/2 phosphorylation in AtT-2 cells are necessary to determine the presence of any functional selectivity. The N4D variant did not affect signalling of any of the other 1 opioid ligands tested, including β-endorphin. The first in vitro studies of N4D signalling reported a threefold increase in β-endorphin affinity for MOPr-N4D in AV-12 cells, as well as a threefold increase in β-endorphin potency for GIRK activation in Xenopus oocytes (Bond et al., 1998). We found no difference for MOPr-N4D in GIRK activation in AtT2 cells, or in AC inhibition or ERK1/2 phosphorylation in CHO cells. Other studies have also failed to find differences in β-endorphin coupling to other signalling pathways in various expression systems (Befort et al., 21; Beyer et al., 24; Kroslak et al., 27). Several studies have investigated the effect of N4D on I Ca channel inhibition, with reports of enhanced DAMGO and morphine signalling (Margas et al., 27; Lopez Soto and Raingo, 212) via N4D, no change in DAMGO signalling (Ramchandani et al., 211), and decreased morphine potency at N4D (Mahmoud et al., 211). We found no significant differences in DAMGO or morphine signalling between -MOPr and N4D-MOPr. Most of the functional studies performed to date have failed to take into account receptor expression and/or receptor reserve, which may contribute to the inconsistency between results. Furthermore, most of the assays were performed under different experimental conditions, making direct comparison between studies difficult (reviewed in Knapman & Connor, 214). In our study, the assays of AC and perk1/2 were performed on CHO cells expressing MOPr- and MOPr-N4D with an isogenic cellular background, similar levels of receptor expression and in very similar experimental conditions, enabling direct comparisons of opioid potency and efficacy to be made between the variants and signalling pathways. It is interesting to note that DAMGO and morphine had similar efficacies to promote ERK phosphorylation in MOPr- cells, but that this efficacy was significantly less than for endomorphin 2 and appeared less than several other ligands such as met-enkephalin and oxycodone. There is little, if any, data examining the efficacy for different MOP receptor ligands activating ERK or comparing efficacy of ERK activation with other effectors. The apparently high efficacy for ERK phosphorylation of endomorphin 2 and oxycodone bears further examination in light of the increasing interest in signalling bias at MOP receptors, particularly as endomorphin-2 has been identified as a significantly biased agonist for recruitment of arrestin3 over G-protein activation (Rivero et al., 212), and either pathway can potentially contribute to ERK activation. Many previous studies and our preliminary time course data indicated that μ-opioid activation of ERK peaks at around 5 min, the time point we chose for our assay. However, it is possible that subtle differences in the time course of ERK activation mean that DAMGO efficacy is slightly underestimated at 5 min compared with some other ligands. There are no functional studies investigating the consequences of the N4D variant on buprenorphine signalling published. However, the consistent loss of buprenorphine effectiveness in several cellular expression systems and distinct signalling pathways suggest that the negative effect of the N4D variant on buprenorphine signalling may be significant in other cell types such as neurons, where MOP receptor is endogenously expressed. In populations where the N4D variant occurs at a high frequency (Mura et al., 213), a significant number of people will be homozygous for the 118G allele; however, the majority of carriers will be heterozygous for the 118A allele. Few clinical studies distinguish between homozygous and heterozygous carriers (e.g. Bond et al., 1998; Bortsov et al., 212; Song et al., 213; Solak et al., 214), and for the few studies that consider both genotypes the data are often contradictory and sample sizes are small (Klepstad et al., 24; Reyes-Gibby et al., 27). There is still considerable uncertainty about the formation of obligate dimers by MOP receptors and other Class A GPCR in native tissue (Herrick-Davis et al., 213; Malik et al., 213), and cells expressing both N4 and D4 receptors could have two populations of receptors independently signalling as monomers or homodimers, or variant receptors could interact and form heterodimers. Functional studies in cells co-expressing both and variant receptors have not been performed, largely due to the technical difficulty of accurately quantifying and expressing equivalent amounts of each variant. In one study, HA-tagged MOPr- and a FLAG-tagged MOP receptor-l85i variant that undergoes endocytosis in response to morphine were co-expressed in HEK293 cells. Both MOPr- and MOPr-L85I internalized in response to morphine, suggesting the formation of functional heterodimers with L85I showing a dominant phenotype (Ravindranathan et al., 29), although alternative explanations are also possible. Conversely, when MOPr- and a non-functional MOPr-R181C variant were co-expressed, MOPr- internalized independently in response to DAMGO, indicating that this variant either fails to form dimers or forms unstable dimers with MOPr- (Ravindranathan et al., 29). These results suggest that the potential for interaction between MOP receptor variants may be dependent on the SNP, and the receptor dynamics resulting from co-expression of MOPr- and MOPr-N4D warrant further investigation. Buprenorphine is commonly prescribed as both an analgesic and as an alternative to methadone for the treatment of opioid dependence, and differences in signalling arising from the N4D variant may be of clinical significance (Virk et al., 29). Most studies investigating differences in opioid requirements for N4D carriers in a clinical setting have not included buprenorphine. For example, a recent large study of European cancer patients investigated the association British Journal of Pharmacology (214)